Reticuloendotheliosis virus (REV), an acute leukemia virus, induces a rapidly lethal lymphoproliferative disease in chickens and transforms avian fibroblasts and hematopoietic cells in vitro. Studies are underway to characterize the integration sites of transforming, replication-defective REV and of REV-A, its non-transforming helper virus. Metaphase chromosomes from an in vivo derived REV-transformed bone marrow cell line have been separated into size classes by zonal centrifugation. DNA transfection experiments have demonstrated that infectious REV-A sequences are found only in the intermediate size class of macrochromosomes. Noninfectious reticuloendotheliosis sequence were detected by hybridization procedures in different size class chromosomes. Complementary DNA probes will be prepared against the unique sequence in the transforming virus (REV) and in the helper virus (REV-A). These probes will be used to locate REV and REV-A sequence in different chromosome fractions obtained from a number of REV-transformed clones. Transfection experiments will be used to locate the integration site containing the infectious helper virus. Transfection experiments using spleen cells will be employed to locate the functional REV transforming sequences in different chromosome fractions. Restriction endonuclease mapping studies conducted with chromosomal DNA containing inactive (noninfection, nontransforming) and active (infectious, transforming) REV-A and REV sequences will define whether integration into a specific site is essential for production of infectious virus and transformation of cells by this acute leukemia virus.